Supplementary Material 1 to “A Self-Replicating Radiation-Shield for Human Deep-Space Exploration: Radiotrophic Fungi can Attenuate Ionizing Radiation aboard the International Space Station” A. Background on Cladosporium sphaerospermum ​ Cladosporium sphaerospermum, a melanized, radiotrophic fungus, characterized chiefly by ​ its ability to utilize ionizing radiation for metabolic functions, was originally discovered in 1886 by 1 Albert Julius Otto Penzig .​ It is known to be saprotroph, xerotolerant and halotolerant; radiotropism, ​ however, was first discovered following the disaster at the Chernobyl nuclear power-plant, when 2 investigating dematiaceous fungi thriving in the surrounding highly radioactive environment :​ fungal ​ samples taken from Chernobyl that were exposed to radiation levels approximately 500-times higher than background level, were observed to grow considerably (up to three times) faster than those not 3 exposed to radiation .​ These organisms continued to grow in the direction of the radiation, another ​ 4 indication for the active pursuit of the radiation as energy-source and thus radiotropism .​ ​ Radiosynthesis is perceived analogous to photosynthesis, except that where the intricacies of photosynthesis have been extensively studied and are well understood, radiosynthesis remains mostly uncharted territory. While the exact mechanisms of radiotropism remain elusive, it has been found that radiation alters the chemical properties of melanin, which leads to increased rates of 5 electron-transfer, allowing for a net energy-gain .​ The pigment melanin may thus act in a manner ​ similar to the chlorophyll in photosynthesis, p​ roviding energy to be used to reduce carbon. Unlike 6 melanocytes in humans, the melanin is crystallized on the cell-walls of the fungi .​ This provides ​ protection of the cells from oxidizing agents generated by different forms of ionizing and non ionizing radiation (ultraviolet light), giving it an evolutionary advantage in extreme natural and non-natural environments on Earth. The expectation is that these properties may also prove advantageous in a 4 Space environment. Therefore, analogous to an existing Earth-based study ,​ C. sphaerospermum was ​ ​ selected as the subject of an experiment on the International Space Station (ISS). B. Methodology and Experiment Absorption Spectrum of Geiger-Counters and Measured Radiation-Levels Ionizing cosmic radiation, or in other words radiation with enough kinetic or electromagnetic energy to strip electrons from an atom, comes in two forms (excluding photons from the classification): wave radiation (electromagnetic waves) and particle radiation, like alpha- and beta-particles, as well as HZE ions. In the electromagnetic spectrum, ionizing (wave-)radiation is characterized by energies ranging from a few hundred eV to about 1 MeV. The PocketGeiger Type5 ​ measures only wave radiation, specifically of X- and gamma-rays (the X100-7 SMD (First Sensor ​ ​ AG, Berlin, Germany) is sensitive at 23°C for gamma-radiation with energies of 3-20 keV, with 7 highest absorbance between 5.5 keV and 10 keV) .​ ​ Based on the total cumulative recorded counts over the 30 days of the experiment a recorded dose equivalent around 1 µSv/h (with a correlation between counts and radiation dose for the ​ PocketGeiger Type5 of 4 CPM ≈ 0.075 ± 0.025 µSv/h)7 was estimated. Given additional shielding due ​ to housing, etc. and considering the narrow absorption spectrum of the Geiger counters, this seems plausible, as it is within one order of magnitude of reported radiation levels on the ISS (≈ 144 µSv/a ≙ 8 16.44 µSv/h ,​ which is to the largest part owed to high-energy nuclei, the most energy-intense fraction ​ 8 of Galactic Cosmic Radiation) .​ ​ Flight Hardware The flight-hardware was packaged as a 2U (double standard-size) CubeLab™ (sealed) ​ module (4”×4”×8”), which has a volume of 103.4 in³, is air-tight, and provides up to 20W of power. The assembled unit is shown in figure S1. Figure S1: Fabricated flight hardware unit, packaged in a 2U Space Tango CubeLab™. ​ ​ Media Courtesy: Space Tango, Inc. The assembled flight-hardware was calibrated and vetted before flight, in particular consistency of the two Geiger counters was confirmed, so that no deviation existed between them. Furthermore, it was ensured that no fungal growth would occur prior to activation (t0) while enroute to ​ ​ the ISS, by means of cold-stow during transport. On orbit the unit was oriented in a way that the Petri dish faced away from Earth. C. On-Orbit Growth Growth of C. sphaerospermum was characterized based on the relative brightness of a ​ representative section in photos of the culture in a Petri dish. These sections (on average 350 by 350 pixels of the fungus-side) were cropped from the rest of the photo, and the average color of each crop was determined in hexadecimal values. The values were then converted to decimal values, yielding a relative brightness-level as a numerical measure for the fungal growth in the Petri dish. The values were standardized to t = 0, normalized to t = 48 h as maximum (cf. supplementary file 2) and plotted ​0 over time, yielding a relative growth curve, as shown in figure S2. Figure S2: Growth curves for C. sphaerospermum on PDA. For the on-orbit experiment (red) the first ​ ​ 48 h are shown in intervals of 3 h, deducted from graphical data (optical density scale normalized to 1 = maximum growth). Due to the few data points for the ground-control (blue), only a qualitative comparison is possible. While for the on-orbit experiment pictures were taken every 30 min., the ground-control was based on cultures from the cold-stow test with one picture taken each day. Further, the ground-control was grown at an ambient temperature of 26°C, opposed to the on-orbit experiment, which equilibrated at 31.5°C. It has been shown though that up to a relative humidity of 76% there is no significant 9 difference in the growth rate of several fungi, cultivated at 20°C and 30°C .​ At 98% relative humidity, ​ the rate of growth is roughly[1] 1.2-times (and never more than 1.3-times) as high at 30°C as compared ​ to 20°C. Since in the current experiment the plates were sealed with parafilm, mass transport of damp can be assumed to be limited and therefore the air inside the plate to be saturated. Over 48 hours this could lead to a difference of 8 hours, which is, however, insignificant in the magnitude of discernible difference between growth times for the ground-control and the flight-experiment as in figure S2. Further, C. sphaerospermum has a reported optimum temperature of 25°C, with an upper limit of 10 ​ 35°C .​ Thus, it becomes more likely that at 31.5°C no growth advantage exists over 26°C. ​ D. Linear (Exponential) Attenuation Background and Definition A linear attenuation coefficient (LAC, symbol “휇”) is a constant, given in per unit thickness ​ ​ 11 of a material and describes the fraction of attenuated incident photons in a monoenergetic beam .​ ​ Therefore, it can be used to determine the probability that ionizing electromagnetic radiation will be scattered by a material, scaled on a linear attenuation index. An LAC is a material-intrinsic property ​ and describes the ability of a material to shield against radiation, measured in change in energy levels per density multiplied by width. It takes into account initial and final radiation levels, as well as the 1 average for the five fungi (A. candidatus: ~ 1.05×, A. flavus: ~ 1.15×, A. fumigatus: ~ 1.24×, P. ​ ​ ​ ​ ​ ​ ​ 9 ​ fellutanum: ~ 1.13×, P. islandicum: ~ 1.3×), as derived from figure 1 of the respective study .​ ​ ​ ​ ​ thickness of the material and its density, to determine the probability that individual atoms within the material will scatter energy waves. Calculations based on LACs were used to interpret the results gathered in the experiment and put them into perspective. Equations for LAC Calculations -(휇/⍴)푥 ​ ​ Eq. 1 I = I0e ​ ​ ​ ​ ​ Eq. 2 휇푚 = ∑퓌풾 풾 ​ ​ ​ ​ ( ρ) where: ● I = intensity at a depth of x cm in keV ​ ​ ​ ​ ● I0 = original intensity in keV ​ ​ ​ ● 푥 = thickness of material ​ ● 퓌풾 = weight fraction of the component material 푖 ​ ​ The estimated dose equivalents based on the counts-per-minute (CPM) averaged over the whole course of the experiment (cf. section B as well as supplementary file 2), were used in the following analysis: ● IControl = 0.916 µSv/h ​ ​ ​ ​ ● IExp = 0.894 µSv/h ​ ​ ​ ​ With the thickness of the fungal lawn 푥 ≈ 0.167 cm, only I0, the ambient radiation levels, ​ ​ ​ inside the lab, remain unknown, in order to be able to determine 휇푚 of C. sphaerospermum (μCsp), ​ ​ ​ ​ ​ ​ according to Eq. 1. Since I0-Control = I0-Fungus, I0-Fungus can be found by plugging IControl, 푥Control, and 휇Control ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ into Eq. 1. The control is constituted of a polystyrene (PS) Petri dish, PDA agar and air, and therefore is composed of three independent linear attenuation coefficients. The linear attenuation coefficient of the air in the Petri dish between the PDA agar and the top lid of the Petri dish is negligible, therefore the working LAC for the Petri dish and PDA as a whole can be calculated as follows. 1. Step 1 – find 휇 ​Control With a density of PS of 1.05 g/cm3 and a 2.2 mm wall strength of the Petri dish, as well as ​ density of the medium of 1.036 g/cm3 (water + 39 g/L PDA) and 13.33 mm average depth, any given ​ 1 cm2 cross section of the control dish consists of 2×0.231 g PS (Petri dish lid and base) and 1.378 g ​ PDA agar, resulting in a total mass of 1.84 g of the 1 cm2 cross section. With linear attenuation ​ coefficients of both PDA and PS retrieved from the NIST-XCOM database12 (for PDA relative ​ percent masses of water, agar, glucose and starch of 96.235%, 1.448%, 1.931% and 0.386% were used, respectively), the cumulative linear attenuation coefficient of the control can be determined with Eq.